Methods for solid-phase synthesis of hydroxylamine compounds and derivatives and combinatorial libraries thereof

ABSTRACT

A novel method for generating hydroxylamine, hydroxamic acid, hydroxyurea, and hydroxylsulfonamide compounds is disclosed. The method involves the nucleophilic attack of an alkoxyamine on a suitable solid phase support. Techniques of combinatorial chemistry can then be applied to the immobilized alkoxyamine to generate a diverse set of compounds. Cleavage of the compounds from the support yields a library of hydroxylamine or hydroxylamine derivative compounds, which can be screened for biological activity (e.g., inhibition of metalloproteases).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of copending U.S.provisional patent application 60/047,468 filed May 23, 1997, and ofcopending U.S. provisional patent application 60/029,788, filed Oct. 28,1996. The contents of both of these applications are hereby incorporatedby reference herein in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not applicable.

TECHNICAL FIELD

[0003] This invention is directed to methods for producing combinatorialchemistry libraries containing hydroxylamines and hydroxylaminederivatives, including hydroxamic acid derivatives, hydroxylureaderivatives, and hydroxysulfonamide derivatives. This invention isfurther directed to synthesis of combinatorial chemistry libraries ofhydroxylamines and hydroxylamine derivatives, including hydroxamic acidderivatives, hydroxylurea derivatives, and hydroxysulfonamidederivatives, using solid-phase techniques. This invention is stillfurther directed to the libraries of hydroxylamines and hydroxylaminederivatives, including hydroxamic acid derivatives, hydroxylureaderivatives, and hydroxysulfonamide derivatives, produced by thesolid-phase synthetic method disclosed. This invention is still furtherdirected to utilizing the libraries of hydroxylamines and hydroxylamicderivatives (including hydroxamic acid derivatives, hydroxylureaderivatives, and hydroxylsulfonamide derivatives) to identify and selectcompounds which bind to, inhibit, or otherwise affect enzymes,receptors, or other biological molecules implicated in disease processes(including disease-related metalloproteases). The hydroxylamines andhydroxylamine derivatives (including hydroxamic acid derivatives,hydroxylurea derivatives, and hydroxylsulfonamide derivatives) thusselected have potential therapeutic value.

BACKGROUND ART

[0004] The techniques of combinatorial chemistry have been increasinglyexploited in the process of drug discovery. Combinatorial chemistryallows for the synthesis of a wide range of compounds with variedmolecular characteristics. Combinatorial synthetic techniques enable thesynthesis of hundreds to millions of distinct chemical compounds in thesame amount of time required to synthesize one or a few compounds byclassical synthetic methods. Subjecting these compounds tohigh-throughput screening allows thousands of compounds to be rapidlytested for desired activity, again saving time expense and effort in thelaboratory.

[0005] Chemical combinatorial libraries are diverse collections ofmolecular compounds. Gordon et al. (1995) Acc. Chem. Res. 29:144-154.These compounds are formed using a multistep synthetic route, wherein aseries of different chemical modules can be inserted at any particularstep in the route. By performing the synthetic route multiple times inparallel, each possible permutation of the chemical modules can beconstructed. The result is the rapid synthesis of hundreds, thousands,or even millions of different structures within a chemical class.

[0006] For several reasons, the initial work in combinatorial libraryconstruction focused on peptide synthesis. Furka et al. (1991) Int. J.Peptide Protein Res. 37:487-493; Houghton et al. (1985) Proc. Natl.Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci.USA 81:3998-4002; Fodor et al. (1991) Science 251:767. The rapidsynthesis of discrete chemical entities is enhanced where the need topurify synthetic intermediates is minimized or eliminated; synthesis ona solid support serves this function. Construction of peptides on asolid support is well-known and well-documented. Obtaining a largenumber of structurally diverse molecules through combinatorial synthesisis furthered where many different chemical modules are readilyavailable; hundreds of natural and unnatural amino acid modules arecommercially available. Finally, many peptides are biologically active,making them interesting as a class to the pharmaceutical industry.

[0007] The scope of combinatorial chemistry libraries has recently beenexpanded beyond peptide synthesis. Polycarbamate and N-substitutedglycine libraries have been synthesized in an attempt to producelibraries containing chemical entities that are similar to peptides instructure, but possess enhanced proteolytic stability, absorption andpharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; andSimon et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9367-9371.Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine librarieshave been synthesized, expanding combinatorial chemistry to includeheterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc.114:10997-10998; Murpy et al. (1995) J. Am. Chem. Soc. 117:7029-7030;and Gordon et al. (1995) Biorg. Medicinal Chem. Lett. 5:47-50.

[0008] Hydroxylamines and their derivatives, including hydroxamic acids,hydroxyl ureas, and hydroxyl sulfonamides, have been the subject of muchresearch focused on their properties as metalloprotease inhibitors.Izquierdo-Martin et al. (1992) J. Am. Chem. Soc. 114:325-33 1; andCushman et al. (1981) Chapter 5 “Specific Inhibitors of ZincMetallopeptidases” in Topics in Molecular Pharmacology (Burgen &Roberts, eds.). Metalloproteases are believed to be involved in thedevelopment of arthritis, tumor angiogenesis, retinopathy, and manyother disease processes.

[0009] U.S. Pat. No. 5,268,384 discloses hydroxamates and hydroxyl ureasused to treat inhibit angiogenesis by inhibiting matrixmetalloproteases. Among metalloproteases disclosed as targets ofinhibitors are collagenases, including human skin fibroblast collagenaseand purulent human sputum collagenase; gelatinases, including human skinfibroblast gelatinase and purulent human sputum gelatinase; andstromelysin. Disclosed disorders amenable to treatment by matrixmetalloprotease (MMP) inhibitors include ocular pathologies such asdiabetic retinopathy and neovascular glaucoma; cancer, includingKaposi's sarcoma, glioblastoma, and angiosarcoma; immune systemdisorders such as rheumatoid arthritis; and skin disorders such aspsoriasis.

[0010] Patent publication WO 96/26918 discloses hydroxamates forinhibiting MMPs. The publication also discusses the inhibition of theproduction or the action of the cytokine tumor necrosis factor (TNF) byhydroxamic acid MMP inhibitors. See also, Mohler et al. Nature370:218-220 (1994); Gearing et al., Nature 370:555-557 (1994); andMcGeehan et al., Nature 370:558-561 (1994). These MMP inhibitors aredescribed as useful for treating inflammatory, infectious, immunologicalor malignant diseases due to their effect on TNF. Among the specificdiseases described are septic shock, hemodynamic shock, malaria,meningitis, fibrotic disease, cachexia, autoimmune diseases, and graftrejection.

[0011] Patent publication WO 96/25156 discloses hydroxamates forinhibiting matrix metalloproteases. The publication also discussesinhibition of production or processing of transforming growth factoralpha (TGF-α) by MMP inhibitors, and describes potential applications ofthe MMP inhibitors in treating inflammation; wound healing, includingscar and keloid formation; diabetic retinopathy; neovascular glaucoma;atherosclerosis; vascular adhesions; systemic lupus erythrematosus;various carcinomas; and other diseases amenable to treatment bymodulating production or processing of TGF-α.

[0012] U.S. Pat. No. 5,552,419 discloses aryl sulfonamido-substitutedhydroxamic acids. The compounds are described as inhibitors ofstromelysin, gelatinase and/or collagenase. Disorders described asamenable to treatment by the hydroxamic acid derivatives areosteoarthritis and rheumatoid arthritis; tissue ulceration; periodontaldisease; bone diseases, including Paget's disease and osteoporosis; HIVinfection; and tumor metastasis, tumor progression or tumor invasion.

[0013] Patent publication EP 423943 describes the use of inhibitors ofcertain matrix metalloproteases, such as collagenases, gelatinases, andstromelysins, as useful for treatment of demyelinating diseases such asmultiple sclerosis and other scleroses; demyelinating peripheralneuropathies; acute disseminated encephalomyelitis; and other neuraldisorders.

[0014] Other hydroxamic acid-based metalloprotease inhibitors aredescribed in the following patent publications: U.S. Pa. Nos. 4,599,361and 5,256,657; European patent publications EP 236872, EP 274453, EP489577, EP 489579, EP 497192, EP 574758; and international PCTapplications WO 90/05716, WO 90/05719, WO 91/02716, WO 92/13831, WO92/22523, WO 93/09090, WO 93/09097, WO 93/20047, WO 93/24449, WO93/24475, WO 94/02446, WO 94/02447, WO 94/21612, WO 94/25434, and WO94/25435.

[0015] Many synthetic routes to produce hydroxylamines have beendeveloped and are well-known in the art (see the above-citedpublications for representative examples). These methods are limited bythe necessity of preparing one compound at a time. Solid-phase synthesisof an immobilized hydroxamate is mentioned in patent application WO96/26918; however, the method used in the application is limited to theUgi reaction described. See also, Strocker et al. Tet. Lett.37:1149-1152 (1996); Keating et al., J. Am. Chem. Soc. 118:2574-2583(1996); and Tempest et al. Angew. Chem. Int. Ed. Engl. 35:640-642(1995), and references therein.

[0016] The invention disclosed herein provides a method forcombinatorial synthesis of hydroxylamines and hydroxylamine derivatives,enabling synthesis of a much greater variety of compounds in arelatively short amount of time.

[0017] All references, publications and patents mentioned herein arehereby incorporated herein in their entirety.

DISCLOSURE OF THE INVENTION

[0018] The present invention provides a method of synthesizing acombinatorial library of hydroxylamines and hydroxylamine derivatives ona solid support, where the first step of the method is the nucleophilicaddition of an alkoxyamine to an appropriate solid support. The alkylgroup forming the alkoxy portion of the alkoxyamine may be a protectinggroup, or may be intended to remain a part of the final compounds. Thesolid support bound alkoxyamine is then derivatized. Followingderivatization, the alkoxyamine derivatives are optionally deprotectedand cleaved from the solid support.

[0019] In another embodiment, the combinatorial libraries aresynthesized by adding an O-alkoxy-protected hydroxylamine-linkerintermediate comprising an O-protected alkoxyamine and a linker group toa solid support bearing an amine group, derivatizing the alkoxyamine,and then optionally deprotecting the alkoxyamine derivatives andcleaving them from the solid support.

[0020] In another embodiment, the method is used to synthesizehydroxylamine and hydroxylamine derivatives selected from the groupconsisting of hydroxylamines, hydroxamic acids, hydroxyl ureas, andhydroxyl sulfonamides.

[0021] The invention also encompasses libraries of the compoundssynthesized by the methods described. These libraries are composed of aplurality of distinct compounds where the classes of compounds include,but are not limited to, hydroxylamines and hydroxylamine derivatives,including hydroxamic acids, hydroxylureas, and hydroxylsulfonamidederivatives. The libraries preferably contain at least about 40, 50, 80,100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000, or 1,000,000distinct compounds, depending on the reactions used for derivatizationat each step and the degree of diversity desired in the library.

[0022] In yet another embodiment, the method is used to synthesizecompounds of the formulas:

[0023] wherein the R groups are independently selected from the groupconsisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclicmoieties as defined herein, as well as amino acid side chains (bothnaturally and non-naturally occurring as defined herein); m, n, and xare integers independently selected from 0 to 12; and y is an integerselected from 0 to 30. The R groups can be attached to asymmetric carbonatoms in either the R-configuration or the S-configuration;additionally, all stereoisomeric and diasteromeric variations of thecompounds and substituents are included in the invention. All protectedderivatives of the compounds and all salts of the compounds are alsoincluded in the invention.

[0024] These libraries include compounds of the form:

L₃—L₂—L₁—NHOH,

[0025] where

[0026] L₃ is selected from the group consisting of

[0027] where the wavy bonds indicate the points of attachment to therest of the molecule. These libraries also include compounds of the form

L₁₂—S(═O)₂—L₁₁—NHOH,

[0028] where

[0029] L₁₂ is selected from the group consisting of

[0030] These libraries also include compounds of the form

L₂₂—S(═O)₂—L₂₁—NHOH

[0031] where L₂₂ is selected from the group consisting of

[0032] and L₂₁ is selected from the group consisting of

[0033] These libraries also include compounds of the form:

[0034] where R₁ is selected from the group consisting of

[0035] and L is selected from the group consisting of

[0036] These libraries also include compounds of the form:

L₂—L₁—NHOH

[0037] where

[0038] L₂ is selected from the group consisting of —H, —C(═O)—CH₃,—C(═O)—OCH₃, and —C(═O)—CH₂—C(═O)—OH; and L1 is selected from the groupconsisting of

[0039] The invention also encompasses O-protected hydroxylaminefunctionalized resins, prepared by displacing a leaving group on a solidsupport by adding an alkoxylamine nucleophile with an alkoxy protectinggroup, resulting in a solid support bound alkoxyamine. Thesealkoxylamine nucleophiles can be O-trityl hydroxylamine,O-(t-butyldimethylsilyl) hydroxylamine, O-allyl hydroxylamine, O-benzylhydroxylamine, O-(4-methoxybenzyl) hydroxylamine, O-(2,4-dimethoxybenzyl) hydroxylamine or O-(2-tetrahydropyranyl)hydroxylamine. The leaving group can be bromide, iodide, or 1 0mesylate.

[0040] The invention also encompasses compounds of the formula

[0041] wherein b is an integer from 1 to 5, X is a leaving groupselected from the group consisting of bromide, iodide, mesylate,tosylate, and p-nitrophenylsulfonate, and RESIN is any amine-bearingresin.

[0042] The invention also encompasses O-protected hydroxylaminefunctionalized resins, prepared by adding an O-protectedhydroxylamine-linker intermediate to a solid support bearing an aminegroup, producing a solid support bound alkoxyamine.

[0043] The invention also encompasses an O-protectedhydroxylamine-linker intermediate suitable for attachment to anamine-bearing resin. Such a compound is made up of an acid-labile linkergroup and an O-protected hydroxylamine.

[0044] The invention also encompasses compounds of the formula

[0045] where b is an integer from 1 to 5, P₁ is a protecting groupselected from the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, J₂ is —H or —moc, and J₁ is —OH or—NH—RESIN, where RESIN is any solid or polymeric support.

[0046] The invention also encompasses derivatized hydroxymethylphenoxyresins and derivatized 2-methoxy-4-alkoxybenzyl alcohol resins, wherethe active hydroxyl group of the resin is replaced with a leaving group.This leaving group can be bromide, iodide, mesylate, tosylate, orp-nitrophenylsulfonate.

[0047] The invention also encompasses compounds of the formula:

[0048] where b is an integer from 1 to 5, P₁ is a protecting groupselected from the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, J₂ is —H or —Fmoc, and J₁ is —OH or—NH—RESIN, where RESIN is any solid or polymeric support.

[0049] The invention also encompasses compounds of the formulas:

[0050] where b is an integer from 1 to 5, and P₁ is a protecting groupselected from the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups.

[0051] The invention also encompasses methods of use of the librariessynthesized by the combinatorial methods to screen for pharmacologicallyactive compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 illustrates a synthetic route to a resin suitable for themethod of synthesis described herein.

[0053]FIG. 2 illustrates a general synthetic method for synthesis ofhydroxamic acid.

[0054]FIG. 3 illustrates a general synthetic method for synthesis ofhydroxylurea.

[0055]FIG. 4 illustrates a general synthetic method for synthesis ofhydroxylamine or hydroxylamine ether.

[0056]FIG. 5 illustrates a synthetic route to an O-allyl protectedhydroxylamine resin.

[0057]FIG. 6 illustrates a synthetic route to an O-allyl protectedhydroxylamine compound.

[0058]FIG. 7 illustrates a synthetic route to a hydroxylamine compoundusing the methods of the invention.

[0059]FIG. 8 illustrates a synthetic route to a hydroxylamine compoundusing the methods of the invention.

[0060]FIG. 9 illustrates a synthetic route to a hydroxylamine compoundusing the methods of the invention.

[0061]FIG. 10 illustrates a synthetic route to a hydroxylamine compoundusing the methods of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0062] The term “bioactive molecule,” as used herein, refers to amolecule that has inhibitory activity. “Inhibitory activity” can bedetermined by inhibition of the interaction between a target and itsrespective substrate(s) or endogenous ligand(s). Target moleculesinclude, but are not limited to, enzymes and receptors. Typically,inhibition is reduced by at least about 15% compared to the interactionof the target and substrate in the absence of the bioactive molecule,where the bioactive molecule is at a solution concentration of 10⁻³molar or lower. Inhibitory activity can also be determined by exhibitionof a dissociation constant of about 10⁻³ of the bioactive molecule withother biological macromolecules, such as DNA, RNA, polysaccharides andproteins not previously included as enzymes or receptors. Preferably,the bioactive molecule has a dissociation constant of about 10⁻⁴ molaror less. More preferably, the molecule has a dissociation constant ofabout 10⁻⁵ molar or less. Most preferably, the molecule has adissociation constant of about 10⁻⁶ molar or less. These macromoleculescan include, but are not limited to, macromolecules derived fromprokaryotic or eukaryotic sources.

[0063] “Hydroxylamine derivatives” include any compounds which arederived from hydroxylamines, including, but not limited to, hydroxylureas, hydroxamic acids, and hydroxyl sulfonamides.

[0064] “Chemical library” or “array” is an intentionally createdcollection of differing molecules which can be prepared syntheticallyand screened for biological activity in a variety of different formats(e.g., libraries of soluble molecules, libraries of molecules bound to asolid support).

[0065] “Alkyl” refers to a cyclic, branched, or straight chain chemicalgroup containing carbon and hydrogen, such as methyl, pentyl, andadamantyl. Alkyl groups can either be unsubstituted or substituted withone or more substituents, e.g., halogen, alkoxy, acyloxy, amino,hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or otherfunctionality that can be suitably blocked, if necessary for purposes ofthe invention, with a protecting group. Alkyl groups can be saturated orunsaturated (e.g., containing —C═C— or —C≡C— subunits), at one orseveral positions. “Heteroalkyl” groups encompass alkyl chains with oneor more N, O, S, or P heteroatoms incorporated into the chain, with theheteroatom bearing none, one, or more than one of the substituentsdescribed above, as well as oxidized forms of the heteroatoms N, S andP. Typically, alkyl groups will comprise 1 to 12 carbon atoms,preferably 1 to 10, and more preferably 1 to 8 carbon atoms.

[0066] “Amino acid” refers to any of the naturally occurring aminoacids, as well as optical isomers (enantiomers and diastereomers),synthetic analogs and derivatives thereof. α-Amino acids comprise acarbon atom to which is bonded an amino group, a carboxyl group, ahydrogen atom, and a distinctive group referred to as a “side chain”.The side chains of naturally occurring amino acids are well known in theart and include, for example, hydrogen (e.g., as in glycine), alkyl(e.g., as in alanine, valine, leucine, isoleucine), substituted alkyl(e.g., as in threonine, serine, methionine, cysteine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl(e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g.,as in tyrosine), and heteroarylalkyl (e.g., as in histidine). See, e.g.,Harper et al. (1977) Review of Physiological Chemistry, 16th Ed., LangeMedical Publications, pp. 21-24. One of skill in the art will appreciatethat the term “amino acid” also includes β- γ-, δ-, and ω-amino acids,and the like and α-imino acids such as proline. As used herein, “aminoacids” includes proline. Non-naturally occurring amino acids are alsoknown in the art, as set forth in, for example, Williams (ed.),Synthesis of Optically Active α-Amino Acids, Pergamon Press (1989);Evans et al., J. Amer. Chem. Soc., 112:4011-4030 (1990); Pu et al., J.Amer. Chem. Soc. 56:1280-1283 (1991); and Williams et al., J. Amer.Chem. Soc., 113:9276-9286 (1991); and all references cited therein.

[0067] “Aryl” or “Ar” refers to a monovalent unsaturated aromaticcarbocyclic group having a single-ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl), which are optionallyunsubstituted or substituted with amino, hydroxyl, lower alkyl, alkoxy,chloro, halo, mercapto, and other substituents.

[0068] “Electron withdrawing group” refers to a substituent that drawselectrons to itself more than a hydrogen atom would if it occupied thesame position in a molecule. Examples of electron withdrawing groupsinclude —NR₂, —COOH, —OR, —SR₂, —F, —COR, —Cl, —SH, —NO₂, —Br, —SR,—SO₂R, —I, —OH, —CN, —C═CR, —COOR, —Ar, —CH CR₂, where R is alkyl, aryl,arylalkyl, or heteroaryl. “Heteroaryl” or “HetAr” refers to a monovalentunsaturated aromatic carbocyclic group having a single ring (e.g.,pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl orbenzothienyl) and having at least one hetero atom, such as N, O, or S,within the ring, optionally unsubstituted or substituted with amino,hydroxyl, alkyl, alkoxy, halo, mercapto, and other substituents.

[0069] “Protecting group” refers to a chemical group that exhibits thefollowing characteristics: 1) reacts selectively with the desiredfunctionality in good yield to give a protected substrate that is stableto the projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield by reagents compatiblewith the other functional group(s) present or generated in suchprojected reactions. Examples of suitable protecting groups can be foundin Greene et al. (1991) Protective Groups in Organic Synthesis, 2nd Ed.(John Wiley & Sons, Inc., New York). Preferred terminal amino protectinggroups include, but are not limited to, benzyloxycarbonyl (CBz),t-butyloxycarbonyl (Boc), t-butyldimethylsilyl (TBDIMS),9-fluorenylmethyloxycarbonyl (Fmoc), or suitable photolabile protectinggroups such as 6-nitroveratryloxy carbonyl (Nvoc), nitropiperonyl,pyrenylmethoxycarbonyl, nitrobenzyl, dimethyl dimethoxybenzil,5-bromo-7-nitroindolinyl, and the like. Preferred hydroxyl protectinggroups include Fmoc, TBDIMS, photolabile protecting groups (such asnitroveratryl oxymethyl ether (Nvom)), Mom (methoxy methyl ether), andMem (methoxy ethoxy methyl ether). Particularly preferred protectinggroups include NPEOC (4-nitrophenethyloxycarbonyl) and NPEOM(4-nitrophenethyloxymethyloxycarbonyl).

[0070] The phrase “distinct compound” is used to refer to compoundswhich are chemically different, except that salts of a compound are notconsidered distinct from the corresponding compound from which the saltis derived. Compounds which differ in molecular formula are definedherein as distinct compounds (except for a compound and its salt).Compounds with the same molecular formula but different bondconnectivities (often referred to as structural isomers), such asleucine and isoleucine, are defined herein as distinct compounds. Acompound and its salt are not defined herein as distinct compounds; forexample, L-valine and L-valine hydrochloride salt are not defined hereinas two distinct compounds. Enantiomers and diastereomers (opticalisomers) are defined herein as distinct compounds; thus, L-valine andD-valine are defined herein as two distinct compounds.

[0071] In the illustrations of libraries that can be made using themethod of the invention, certain groups are drawn with wavy bondsindicating their points of attachment to the rest of the molecule. Forgroups written as text (e.g., —CH₃), the dash indicates the point ofattachment to the rest of the molecule. For groups which have a bondwhich intersects a wavy line, the intersection point indicates the pointof attachment to the rest of the molecule. Thus, for compounds of theform L₃—L₂—LI—NHOH, when L₃, L₂, and L₁ are

[0072] respectively, the compound

[0073] is

[0074] For compounds of the form L₁₂—S(═O)₂—L₁₁—NHOH, when L₁₂ and L₁₁are

[0075] respectively, the compound is

[0076] For compounds of the form L₁₂—S(═O)₂—L₁₁—NHOH, when L₁₂ and L₁₁are

[0077] respectively, and Z is

[0078] (where the Z group has a bond which intersects a wavy line), thecompound is

[0079] For compounds of the form

[0080] when R₁ is

[0081] and L is

[0082] the compound is

Abbreviations

[0083] The following abbreviations are used:

[0084] AcOH, HOAc=acetic acid

[0085] Ac₂O=acetic anhydride

[0086] BOC, Boc=t-butyloxycarbonyl

[0087] DCM=dichloromethane

[0088] DIC=diisopropylcarbodiimide

[0089] DIAD=diisopropylazodicarboxylate

[0090] DIEA=diisopropylethylamine

[0091] DMF=dimethylformamide

[0092] Et=ethyl

[0093] EtOAc=ethyl acetate

[0094] Fmoc, FMOC=9-fluorenylmethyloxycarbonyl

[0095] HATU=O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

[0096] HBTU=O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

[0097] HHMPA=(4-hydroxymethyl-3-methoxyphenoxy)-alkanoic acid

[0098] HMP resin=hydroxymethylphenoxy resin

[0099] HOAt=1-hydroxy-7-azabenzotriazole

[0100] HOBt=1-hydroxybenzotriazole

[0101] Me=methyl

[0102] Mem=methoxy ethoxy methyl ether

[0103] MeOH=methanol

[0104] MMP=matrix metalloproteinase

[0105] Mom=methoxy methyl ether

[0106] NMM=N-methyl morpholine

[0107] NPEOC=4-nitrophenethyloxycarbonyl

[0108] NPEOM=4-nitrophenethylmethyloxycarbonyl

[0109] NVOC=6-nitroveratryloxycarbonyl

[0110] NVOM=nitroveratryloxymethyl ether

[0111] PEG-PS resins or PS-PEG resin=polyethylene glycol-polystyrenegraft copolymer resins

[0112] PyBOP=benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate

[0113] RT=room temperature

[0114] TBP=tributylphosphate

[0115] TBS, TBDIMS=t-butyldimethylsilyl

[0116] tBu=t-butyl

[0117] TES=triethylsilane

[0118] TFA=trifluoroacetic acid

[0119] TGS resin=TENTAGEL S resin

[0120] TGS NH₂ resin=TENTAGEL S NH₂ resin

[0121] THF=tetrahydrofuran

[0122] THP=2-tetrahydropyranyl

[0123] TMAD=N,N′,N′-tetramethylazodicarboxamide(1,1′-Azobis(N,N-dimethylformamide))

[0124] TMOF=trimethylorthoformate

[0125] TPP=triphenyl phosphine

[0126] TsCl=tosyl chloride

[0127] Trt=trityl

General Resins

[0128] Common resins used for peptide synthesis and adaptable forcombinatorial synthesis include, but are not limited to,hydroxymethylphenoxy (HMP) resin; 2-methoxy-4-alkoxybenzyl alcoholresin, and polystyrene-polyethylene glycol graft copolymer-derivedresins.

[0129] Wang resin is the trade name for hydroxymethylphenoxy (HMP)resin, also called p-alkoxybenzyl alcohol resin or p-benzyloxybenzylalcohol resin. This resin is described in Wang (1973), J. Am. Chem. Soc.95: 1328. The resin has the following structure:

[0130] The polystyrene backbone can be crosslinked by 0.5-2.0%divinylbenzene, typically about 1% divinylbenzene.

[0131] 2-methoxy-4-alkoxybenzyl alcohol resin is a resin which is highlylabile to acid cleavage. It is sold under the trademark SASRIN resin(SASRIN is a registered trademark of Bachem Bioscience, King of Prussia,PA). This resin is described in Mergler et al., U.S. Pat. No. 4,831,084and has the following structure:

[0132] The polystyrene backbone can be crosslinked by about 0.5-2.0%divinylbenzene.

[0133] Resins comprising polyethylene glycol grafted onto polystyrenehave come into wide use recently as supports for solid-phase synthesis.These resins will be generically referred to as polyethyleneglycol-polystyrene graft copolymer resins, or PEG-PS resins One suchPEG-PS resin is sold under the trademark TENTAGEL. Derivatives of thisresin are sold under the trademark NOVASYN. TENTAGEL is a registeredtrademark of Rapp Polymere (Tubingen, Germany). NOVASYN is a registeredtrademark of Calbiochem-Novabiochem (San Diego, Calif.). The TENTAGELpolystyrene-polyethylene glycol graft copolymer resin is described inBayer et al., U.S. Pat. No. 4,908,405. It has the following structure:

[0134] The PEG-PS resins can be generically written as

[0135] For the PEG-PS polymer TENTAGEL, M is —CH₂—. Other linkers Mbetween the polyethylene glycol (PEG) polymer and the polystyrenepolymer are possible; one such linker is—CH₂CH₂—NH—(C═O)—CH═CH—(C═O)—NH—, illustrated in Barany et al., EP687691. The term “PEG-PS resin” or “polyethylene glycol-polystyrenegraft copolymer resin” is intended to include, but not be limited to,both TENTAGEL and the polyethylene glycol-polystyrene graft copolymerresins of EP 687691. The polystyrene backbone can be crosslinked byabout 0.5-10.0% divinylbenzene, preferably about 1-2% divinylbenzene.The polyethylene glycol chains can have a molecular weight of about 500to about 40,000, corresponding to a value for n of about 10 to 1,000. Apreferred range for n is about 65 to 75; a still more preferable valuefor n is 68. X can be selected from many functional groups, including,but not limited to, —Br, —NH₂, —SH, —COOH, and —OH. These resins arereferred to, respectively, as bromo-terminated polyethyleneglycol-polystyrene graft copolymer resin (Br-PEG-PS resin orbromo-PEG-PS resin); amino-terminated polyethylene glycol-polystyrenegraft copolymer resin (amino-PEG-PS resin or NH₂-PEG-PS resin) (TENTAGELS NH₂ resin is an example of an amino-PEG-PS resin); thiol-terminatedpolyethylene glycol-polystyrene graft copolymer resin (thiol-PEG-PSresin or HS-PEG-PS resin); carboxy-terminated polyethyleneglycol-polystyrene graft copolymer resin (carboxy-PEG-PS resin); andhydroxy-terminated polyethylene glycol-polystyrene graft copolymer resin(hydroxy-PEG-PS resin or HO-PEG-PS resin). Resins can terminate in moreelaborate groups as well, often by derivatizing the resins alreadydescribed. TENTAGEL resins are commercially available from Novabiochem(San Diego, Calif.) and Peptides International (Louisville, Ky.).

Resins for Combinatorial Synthesis

[0136] A solid phase support suitable for preparing combinatoriallibraries of hydroxylamine and hydroxylamine derivative compounds mustanchor the synthetic intermediates stably during the chemical stepsrequired to assemble the compounds on the support. Once synthesis of thecompounds is completed, they must be capable of being cleaved from thesupport under conditions which do not have a significantly deleteriouseffect on the compounds. Solid phase supports which meet these criteriahave been derived from hydroxymethylphenoxy (HMP) resin (Wang, S. S., J.Am. Chem. Soc. 95:1328 (1973)) (available from Advanced ChemTech,Louisville, Ky.) and TENTAGEL S AC resin (Florsheimer et al., Peptides1990: Proceedings of the 21 st European Peptide Symposium (Giralt andAndreu, eds.); Leiden: Escom, 1991, p. 131; available from RappPolymere, Tubingen, Germany, and Advanced ChemTech, Louisville, Ky.).SASRIN resin (Bachem Bioscience, King of Prussia, Pa.) can also bemodified in a similar manner to HMP and TENTAGEL S AC resins and used inthe method of the invention.

[0137]FIG. 1 shows various methods of preparing resins, and subsequentlyimmobilizing a hydroxylamine derivative onto the resin. See Ngu andPatel, Tet. Lett. 38:973 (1997). In FIG. 1, a starting resin 4 (HMP orTENTAGEL S AC resin) is functionalized to replace the active hydroxylgroup with a leaving group X, where X may be bromine, chlorine, oriodine (see Table 1 below). This functionalized resin 1 can then bereacted with a hydroxylamine of the form 2 (where R₁ is an alkyl groupor protecting group as defined herein) to form an alkoxylamine resin 3,which can be further derivatized. Of these resins functionalized with aleaving group, bromomethylphenoxy resin (brominated HMP resin) ispreferred for its high yield from parent resin (99%) and suitability forfurther synthetic steps. In instances where milder cleavage conditionsare required, brominated or iodinated TENTAGEL S AC resin (cleavablewith 5% trifluoroacetic acid) is preferred. Resins which can be used inthe invention, are (but are not limited to) as follows:

[0138] where HHMPA refers to the reagent

[0139] (4-hydroxymethyl-3-methoxyphenoxy)-alkanoic acid, where b is aninteger from 1 to 5. An example of this type of resin is TENTAGEL S ACresin. TENTAGEL S AC resin is used in the examples below; however, anyHHMPA—NH2-PEG-PS type resin can be used in an analogous fashion.Utilizing the reagents described in Table 1 gives resins of the type:

[0140] Conversion of the original HMP or TENTAGEL S AC resin to thederivatized form was assayed by reacting n-butylamine with thederivatized resin, then washing away unreacted amine. The resin was thenreacted with 2-naphthalenesulfonyl chloride. Resins on which the X groupwas displaced by nucleophilic attack of the amine yieldedn-butyl-(2-naphthyl)-sulfonamide upon cleavage of the resin withtrifluoroacetic acid. Resins not converted to the derivatized form, orunstable after conversion and reverted back to the original resin,yielded 2-naphthalenesulfonic acid upon cleavage. The yield ofsulfonylated amine (and hence of stable derivatized resin in the schemedepicted above) is summarized in Table 1, along with the reagents usedfor the derivatization of the resins. TABLE 1 Reagents for DerivatizingHMP and TENTAGEL S AC Resins Yield of X Yield of Derivatived (leavingDerivatived Amine from group on Amine from Modified modified ModifiedTENTAGEL S Reagents resin) HMP resin AC resin Ph₃PBr₂ or Ph₃P/CBr₄ Br99% 87% Ph₃PI₂ or Ph₃P/ I 93% 79% diisopropyl azodicarboxylate/ CH₃Imethanesulfonyl chloride/ mesyl 95% not assayed N-methyl morpholinetoluenesulfonyl chloride/ tosyl  0% not assayed N-methyl morpholine4-nitrobenzenesulfonyl nosyl  0% not assayed chloride/ N-methylmorpholine

[0141] An alternative route to producing a resin suitable forcombinatorial synthesis is provided by coupling a hydroxylamine compoundto a linker in solution, then attaching the linker to an appropriateresin. The scheme below illustrates the attachment of a linker (Sharmaet al., J. Org. Chem. 58:4993 (1993); Albericio et al., Tet. Lett.32:1015 (1991);

[0142] Albericio et al., J. Org. Chem. 55:3730 (1990) to the O-protectedhydroxylamine compound NH₂—O—P₁, where P₁ is a protecting group.Examples of O-protected hydroxylamines include, but are not limited to,NH₂—O—THP (O-(2-tetrahydropyranyl) hydroxylamine), O-tritylhydroxylamine (Trt-O—NH₂), O-(t-butyldimethylsilyl) hydroxylamine(TBS-O—NH₂), O-(allyl) hydroxylarnine (allyl-O—NH₂), O-benzylhydroxylamine (PhCH₂—O—NH₂), O-(4-methoxybenzyl) hydroxylamine(4MeOPhCH₂—O—NH₂), O-(2, 4-dimethoxybenzyl) hydroxylamine (2,4-diMeOPhCH₂-O—NH₂), and other protecting groups compatible with thechemical steps used in the synthesis. Specific examples of using theprotecting groups THP and allyl are illustrated below.

Schemes for Coupling a Linking Group to the Protected Hydroxylamine

[0143]

[0144] where

[0145] P1 is a protecting group as described above, and b is an integerfrom 1 to 5

[0146] The following scheme illustrates the synthesis of the protectedhydroxylamine-linker using THP as the protected

[0147] where

[0148] b is an integer ranging from 1 to 5.

[0149] The following scheme illustrates the synthesis of the protectedhydroxylamine-linker using allyl as the protecting group:

[0150] This protected hydroxylamine-linker intermediate can then becoupled to any of a variety of amine resins and then used forsolid-phase combinatorial synthesis.

[0151] The following scheme depicts coupling of the protectedhydroxylamine-linker to the resin, with P₁ a protecting group asindicated above:

[0152] where

[0153] b is an integer from 1 to 5.

[0154] The following scheme depicts coupling of the protectedhydroxylamine-linker to the resin, with THP as the protecting group:

[0155] where

[0156] b is an integer from 1 to 5.

[0157] The following scheme depicts coupling of the protectedhydroxylamine-linker to the resin, with allyl as the protecting group:

[0158] where

[0159] b is an integer ranging from 1 to 5. Examples of amine resins towhich the protected hydroxylamine-linker intermediate can be coupledinclude, but are not limited to, amino-PEG-PS resins, TENTAGEL S NH₂resin, benzhydrylamine and p-methylbenzhydrylamine resins,aminomethylated polystyrene resin, and other resins bearing an aminegroup.

General Synthesis of Hydroxylamines and Hydroxylamine Derivatives,including Hydroxamic Acids, Hydroxylureas, and Hydroxylsulfonamides

[0160] Hydroxylamine or hydroxylamine derivatives can be coupled to thederivatized resins for further chemical transformations, as depicted inFIG. 1. Coupling of O-protected hydroxylamines prevents side reactionsfrom occurring at the oxygen atom of the resin-bound hydroxylamine orhydroxylamine derivative during synthesis; the O-protecting group can beremoved at the end of synthesis, either before, during, or after finalcleavage of the compounds from the resin. FIG. 2 illustrates thesynthesis of a hydroxamic acid derivative, and also illustrates removalof the O-protecting group either before or after synthesis. In FIG. 2,the alkoxyamine resin compound 3 is reacted with a carboxylic acid 5(where R₂, R₃ and R₄ are independently selected from the groupconsisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclicmoieties as defined above, as well as naturally-occurring andnon-naturally-occurring amino acid side chains) to produce a hydroxamicacid-resin compound 6. The R₁ group can function as an O-protectinggroup during the synthesis, and can be removed from thehydroxamate-resin complex to form the deprotected hydroxamate-resincomplex 9; the deprotected complex is then cleaved to form the finalproduct 8. Alternatively, the O-protected hydroxamate can be cleavedfrom the resin to yield the O-protected hydroxamate 7, which can then bedeprotected to form the final product 8. Finally, the hydroxylamineether 7 can be the desired product; in other words, R₁ functions notonly as a blocking group during the synthesis, but is a desired part ofthe final compound, in which case it is not removed after cleavage ofthe hydroxamate from the resin. Representative O-protected hydroxylaminecompounds include, but are not limited to, O-trityl hydroxylamine(Trt-ONH₂), O-(t-butyldimethylsilyl) hydroxylamine (TBS-ONH₂), O-(allyl)hydroxylamine (allyl-ONH₂), O-benzyl hydroxylamine (PhCH₂—O NH₂),O-(4-methoxybenzyl) hydroxylamine (4MeOPhCH₂—O—NH₂), O-(2,4-dimethoxybenzyl) hydroxylamine (2, 4-diMeOPhCH₂—O—NH₂), andO-(2-tetrahydropyranyl) hydroxylamine (THP-ONH₂ or Thp-ONH₂). PreferredO-protecting groups are benzyl (cleaved by hydrogenolysis) and trityl(cleaved by trifluoroacetic acid).

[0161]FIG. 3 represents a general method for synthesizing hydroxylureacompounds, starting with an immobilized alkoxyamine 3 and utilizing anisocyanate compound. After synthesis is complete, cleavage anddeprotection of the compound yields the hydroxyl urea 20, where R₂ isselected from the group consisting of H, alkyl, heteroalkyl, aryl,heteroaryl, and heterocyclic moieties as defined above, including alkylgroups substituted with naturally-occurring and non-naturally-occurringamino acid side chains.

[0162]FIG. 4 represents a general method for synthesizinghydroxylamines, including hydroxylamine ethers, starting with animmobilized alkoxyamine 3 and utilizing an alkylating agent. Aftersynthesis is complete and the compound is cleaved from the resin, R₁ canbe removed (if R₁ is a protecting group) to yield the hydroxylamine.Alternatively, if the hydroxylamine ether is desired (i.e., R₁ forms apart of the desired product), then the synthesis is complete aftercleavage from the resin. R₂ is selected from the group consisting of H,alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties asdefined above, including alkyl groups substituted withnaturally-occurring and non-naturally-occurring amino acid side chains.

[0163] Purification of the compounds generated in the invention can beaccomplished by any of the methods of purification well-known in the artof organic synthesis, including but not limited to, recrystallization,column chromatography, flash chromatography, thin-layer chromatography,partitioning between immiscible solvents, HPLC, and affinitychromatography. Enantiomers and diastereomers can be resolved by methodswell-known in the art of organic synthesis, including (but not limitedto) chromatography on chiral resolving material and enzymaticresolution.

[0164] Generation of the Combinatorial Libraries

[0165] The general structures described above can take a wide variety offorms; substitution of various groups at any of the variable positionsyields a plethora of compounds. The power of combinatorial synthesis isreadily exploited by introducing a mixture of reagents at each stepwhere a variable substituent is possible in the structure. For example,if an N-alkylated hydroxy amine is desired, the immobilizedN-alkoxyamine on the resin can be reacted with a mixture of alkylchlorides (for example butyl chloride, 2-chloropropane, and benzylchloride). The reagents can be provided in concentrations inverselyproportional to their rates of reaction with the immobilizedintermediate, so that approximately equal amounts of the variouscomponents of the combinatorial mixture are produced. The reaction ratescan be assayed by techniques well known in the art. One such assayinvolves introducing a single substituent at each of the variable steps,except at the step where coupling rates are to be assayed. At that step,an equimolar mixture of the various reagents which introduce the variedsubstituents can be provided. The intermediates produced after that stepcan be cleaved from the resin, and separated and analyzed on a GC/MS orLC/MS apparatus, or using other appropriate analytical instruments ormethods. This will yield information about the relative coupling ratesof the reagents used. This method is a generalization of the method forassaying reaction rates of reagents for introducing amino acids inpeptide synthesis, provided by Rutter et al. in U.S. Patent No.5,010,175.

[0166] Instead of mixing several reagents at one step as describedabove, combinatorial libraries can also be prepared by using the “splitand pool” protocol described by Furka et al., Int. J. Pept. Prot. Res.37:487 (1991). In this method the total number of reactions grows in anadditive fashion with the number of steps, but the number of compoundsprepared grows in a multiplicative fashion.

[0167] Finally, a combinatorial library can also be prepared byperforming parallel synthesis, where compounds are prepared in parallelas discrete compounds or as small pools of compounds.

[0168] All of these methods for generating combinatorial libraries ofcompounds can be performed in automated, semi-automated, or manualprotocols and techniques, according to methods well known in the art.

[0169] Cleavage of Compounds from the Resin

[0170] Cleavage of the compounds from the derivatized HMP or TENTAGEL SAC resins is accomplished under acidic conditions. Preferably, 95-100%trifluoroacetic acid is used to cleave compounds attached to the HMPresin. A small portion (typically 5% or less) of the cleavage solutioncan be composed of scavenger compounds, the purpose of which is to trapcarbocations released during the cleavage process to prevent the cationsfrom reacting with the desired products. The chemical composition of theproducts and their sensitivity to alkylation by carbocations willdetermine the appropriate scavenger or scavengers. Such scavengercompounds are well-known in the field of peptide synthesis and includesubstances such as thiols (e.g. 1,2-ethanedithiol), phenols,trialkylsilyl compounds, anisole, thioanisole, water, and sulfides(e.g., methyl ethyl sulfide). In appropriate cases it may be desirableto reduce the amount of trifluoroacetic acid below 95% and increase theproportion of scavengers used accordingly. In cleaving compounds fromTENTAGEL S AC-derived resins, typically a solution ranging from 5%-50%trifluoroacetic acid in dichloromethane is used, with the optionaladdition of scavengers such as those indicated above.

[0171] Use of resins incorporating photolabile linkers allows cleavageto be performed by photolysis. Photolabile resins have been described inInternational Patent Application WO 96/00378; in Holmes et al., (1995)J. Org. Chem. 60: 2318; and in Holmes et al., Peptides: ChemistryStructure and Biology. Proc. 14th American Peptide Symposium, MayflowerScientific, 1995, p. 44. Such resins are commercially available fromNovabiochem (San Diego, Calif.). Particularly suitable resins arederived from hydroxymethyl-photolinker AM resin and thehydroxymethyl-photolinker NOVASYN TG resin. (The graft polymer composedof polystyrene and polyethylene glycol is sold under the trademarkNOVASYN TG resin.) The hydroxymethyl group of these resins arederivatized to form bromomethyl resins by using the synthetic protocolsdescribed above under “Resins” and in Example 1 used to converthydroxyrethylphenoxy resin to bromomethylphenoxy resin. The compoundsare synthesized on the support in the same fashion as for thebromomethylphenoxy resin, by displacing the bromide leaving group withan alkoxylamine nucleophile to produce a solid support boundalkoxylamine, as depicted below:

[0172] where

[0173] b is an integer from 1 to 5, P₁ is a protecting group selectedfrom the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, J₂ is -H or -Fmoc, and J₁ is —OH or—NH-RESIN, where RESIN is any solid or polymeric support. Typically,RESIN is a polystyrene-type resin or a NOVASYN TG-type resin. (The graftpolymer composed of polystyrene and polyethylene glycol is sold underthe trademark NOVASYN TG resin.)

[0174] The compounds are derivatized in an analogous manner as in theexamples. The compounds can then be cleaved from the resin using thephotolysis conditions described in the references cited above. Oneexemplary protocol illustrated in WO 96/00378 is the suspension of 2-20mg of resin in pH 7.4 aqueous buffer solution, followed by irradiationwith a 500 W Hg ARC lamp filtered by a 350-450 nm dichroic mirror at a10 mW/cm² power level measured at 365 nm. Irradiation time can be onehour or until satisfactory yield of cleaved compound is detected.

Screening

[0175] The present invention is directed toward the generation oflibraries of hydroxylamines and hydroxylamine derivatives. Theselibraries are used to select one or more hydroxylamine or hydroxylaminederivative species that demonstrate a specific interaction with atargeted enzyme or receptor. An enzyme or receptor is targeted when itis believed that the enzyme or receptor is of importance in themodulation of a disease. Examples of disease states for whichhydroxylamine or hydroxylamine derivative libraries can be screenedinclude, but are not limited to, tumor growth and angiogenesis,arthritis, connective tissue disorders, inflammatory diseases, andretinopathies.

[0176] Several methods have been developed in recent years to screenlibraries of compounds to identify the compounds having the desiredcharacteristics. Typically, where a compound exhibits a dissociationconstant of 10⁻⁶ or less when combined with the targeted enzyme orreceptor, the compound is thought to demonstrate a specific interactionwith the enzyme or receptor. Methods for isolating library compoundspecies that demonstrate desirable affinity for a receptor or enzyme arewell-known in the art.

[0177] For example, an enzyme solution can be mixed with a solution ofthe compounds of a particular combinatorial library under conditionsfavorable to enzyme-ligand binding. Binding of library compounds to theenzyme can be detected by any of the numerous enzyme inhibition assayswhich are well known in the art. Compounds which are bound to the enzymecan be readily separated from compounds which remain free in solution byapplying the solution to a column such as a Sephadex G-25 gel filtrationcolumn. Free enzyme and enzyme-ligand complex will pass through thecolumn quickly, while free library compounds will be retarded in theirprogress through the column. The mixture of enzyme-ligand complex andfree enzyme can then be treated with a powerful denaturing agent, suchas guanidinium hydrochloride or urea, to cause release of the ligandfrom the enzyme. The solution can then be injected onto an HPLC column,for example, a Vydac C-4 reverse-phase column, eluted with a gradient ofwater and acetonitrile ranging from 0% acetonitrile to 80% acetonitrile.Diode array detection can provide discrimination of the compounds of thecombinatorial library from the enzyme. The compound peaks can thencollected and subjected to mass spectrometry for identification.

[0178] An alternate manner of identifying compounds that inhibit anenzyme is to divide the library into separate sublibraries where onestep in the synthesis is unique to each sublibrary. To generate acombinatorial library, reactants are mixed together during a step togenerate a wide mixture of compounds. At a certain step in thesynthesis, however, the resin bearing the synthetic intermediates can bedivided into several portions, with each portion then undergoing aunique transformation. The resin portions are then (separately)subjected to the rest of the synthetic steps in the combinatorialsynthetic method. Each individual resin portion thus constitutes aseparate sublibrary. When testing the compounds, if a given sublibraryshows more activity than the other sublibraries, the unique step of thatsublibrary can then be held fixed. The sublibrary then becomes the newlibrary, with that step fixed, and forms the basis for another round ofsublibrary synthesis, where a different step in the synthesis isoptimized. This procedure can be executed at each step until a finalcompound is arrived at. The aforementioned method is the generalizationof the method described in Geysen, WO 86/00991, for determining peptide“mimotopes,” to the synthetic method of this invention.

[0179] While finding a compound that inhibits an enzyme is most readilyperformed with free compound in solution, the compounds can also bescreened while still bound to the resin used for synthesis. In someapplications, this may be the preferable mode of finding compounds withthe desired characteristics. For example, if a compound which binds to aspecific antibody is desired, the resin-bound library of compounds canbe contacted with an antibody solution under conditions favoring astable antibody-compound-resin complex. A fluorescently labeled secondantibody which binds to the constant region of the first antibody canthen be contacted with the antibody-compound-resin complex. This willallow identification of a specific bead as carrying the compound whichis recognized by the first antibody binding site. The bead can then bephysically removed from the resin mixture and subjected to mass spectralanalysis. If the synthesis has been conducted in a manner such that onlyone compound is likely to be synthesized on a particular bead, then thebinding compound has been identified. If the synthesis has been carriedout so that many compounds are present on a single bead, the informationderived from analysis can be utilized to narrow the synthetic choicesfor the next round of synthesis and identification.

[0180] The enzyme, antibody, or receptor target need not be in solutioneither. Antibody or enzyme can be immobilized on a column. The libraryof compounds can then be passed over the column, resulting in theretention of strongly binding compounds on the column afterweaker-binding and non-binding compounds are washed away. The column canthen be washed under conditions that dissociate protein-ligand binding,which will remove the compounds retained in the initial step. Thesecompounds can then be analyzed, and synthesized separately in quantityfor further testing. Similarly, cells expressing cell surface receptorscan be contacted with a solution of library compounds. The cells bearingbound compounds can be readily separated from the solution containingnon-binding compounds. The cells can then be washed with a solutionwhich will dissociate the bound ligand from the cell surface receptor.Again, the cells can be separated from the solution, and the solutionwhich now contains the ligands bound in the initial step can beanalyzed.

[0181] Assays appropriate for measuring the inhibition or modulation ofthe activity of biological molecules (such as enzymes) by the compoundsof the invention can be found in the following publications: Patel etal., J. Med. Chem. 39: 4197-4210 (1996); Methods in Enzymology Vol. 248,“Proteolytic Enzymes: Aspartic and Metallo Peptidases,” (Alan J.Barrett, ed.), New York: Academic Press, 1995, Chapters 1-6 and 13-51;Methods in Enzymology Vol. 80, New York: Academic Press, 1981, Chapters52 and 53; Cawston et al., Biochem. J. 195:159-165 (1981); Sellers etal., Biochem. J. 171:493-496 (1978); and Cawston et al., Anal. Biochem.99:340-345 (1979). The patent publications mentioned in the BackgroundArt section above also contain useful assays which can be used todetermine the effects which the compounds of the invention have onbiological molecules.

[0182] Pharmacological Applications Those compounds selected as havingpharmacological activity can be useful as drugs for the treatment ofdisease states in mammals, including humans. Such drugs comprisecompounds of the invention in amounts and formulations appropriate fortherapeutic effect in any of the diseases amenable to such treatment.The drugs also comprise any pharmaceutically acceptable salt of thecompounds, as well as any carrier or excipient appropriate for the drug.Carriers and excipients well known in the art can be used, such as thosedisclosed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. The compounds of the invention, or salts thereof,can be formulated in such a manner as to be administered orally;nasally; parenterally (e.g. intravenous and intramuscularly, as asolution); in medicaments for rectal or vaginal application; inmedicaments for application to the skin and mucous membranes (e.g. assolutions, lotions, emulsions, salves, plasters, etc.); and inmedicaments for topical application to the eyes. The compounds can alsobe administered in liposome formulations. The compounds of the inventioncan also be administered as prodrugs, where the prodrug administeredundergoes biotransformation in the treated mammal to a form which isbiologicaly active.

[0183] The following examples are provided as illustrations of themethods described, and are not intended to limit the invention in anyway.

EXAMPLES Example 1

[0184]

Preparation of Bromomethylphenoxy Resin (Brominated HMP Resin).

[0185] 1.42 g PPh₃Br₂ (3 eq.) in 15 ml DCM was added, in 3 equalportions, to a suspension of 1 g of HMP resin (hydroxymethylphenoxyresin) (1 eq.) in 10 ml DCM at room temperature under argon. Afterstirring for 3 hours at room temperature, the reaction mixture wasfiltered, the resin was washed with DCM (4×15 ml), and dried undervacuum to give 1.066 g bromomethylphenoxy resin (99% yield).

[0186] Should a more acid-labile resin be desired, TENTAGEL S AC resincan be employed instead of HMP resin. Compounds immobilized on TENTAGELS AC resin can be cleaved with 5% trifluoroacetic acid (TFA) solution indichloromethane, instead of the higher concentration of TFA used belowin Example 4 with the HMP resin immobilized compounds.

[0187] The corresponding iodo resins can be prepared by using PPh₃I₂ inplace of PPh₃Br₂ in Example 1, above.

Example 2

[0188]

Preparation of O-benzylhydroxylamine-resin.

[0189] O-benzylhydroxylamine was prepared by partitioning 1.0 g ofO-benzylhydroxylamine hydrochloride between 20 ml of ethyl acetate and20 ml of saturated aqueous K₂CO₃ solution. The ethyl acetate layer wasdried and concentrated to give O-benzylhydroxylamine.O-benzylhydroxylamine (2 ml of a 0.5 M solution in dimethylformamide(DMF)) was added to 200 mg of the bromomethylphenoxy resin of Example 1.The reaction mixture was shaken for 24 hours at room temperature. Theresulting alkoxyamine resin was then washed with methanol (3×2 ml) anddichloromethane (3×2 ml).

Example 3

[0190]

[0191] 1 ml of a 0.6 M solution of 2,6-di-t-butyl-4-methylpyridine inDMF and 1 ml of a 0.5 M solution of hydrocinnamoyl chloride(3-phenylpropionyl chloride) was added to the benzylhydroxylamine resinof Example 2. The reaction was stirred at 40° C. for 18 hours. The resinwas washed with methanol (3×2 ml) and dichloromethane (3×2 ml).

Example 4

[0192]

[0193] The immobilized intermediate synthesized in Example 3 was placedin a flask and 10 microliters water and 1 ml trifluoroacetic acid wereadded to the resin. The resin was shaken for 30 minutes. The solutionwas filtered and the filtrate was concentrated by evaporation and driedunder vacuum to yield the compound above. ¹H NMR (300 MHz, CDCl₃): 2.18(t, 2H); 2.97 (t, 2H); 4.83 (s, 2H); 7.15 (m, 10H); 7.78 (s, 1H).

Example 5

[0194]

[0195] The product of Example 4 was dissolved in 10 ml of methanol. 20mg of 10% Pd/C was added and the mixture was stirred under a hydrogenatmosphere for 5 hours at room temperature. The palladium catalyst wasfiltered off through a layer of Celite, and the filtrate wasconcentrated under vacuum to give the hydroxamic acid derivativedepicted above. ¹H NMR (300 MHz, CDCl₃): 2.23 (t, 2H); 2.97 (t, 2H);7.15 (m, 5H); 7.22 (s, 1H)

Example 6

[0196]

Synthesis ofN-hydroxy-2(R)-[[4-methoxybenzenesulfonyl](3-picolyl)amino]-3-methylbutanamide.

[0197] The solution synthesis of the title compound is described in EP606046. The solid-phase synthesis proceeds as follows:

[0198] Step 1. The O-benzylhydroxylamine resin product of Example 2 (1eq.) is reacted with Fmoc-D-Valine (3 eq.),O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)(3 eq.), N-hydroxybenzotriazole (HOBt) (3 eq.), andN,N-diisopropylethylamine (DIEA) (4.5 eq.) in DMF for 3 hours. Thereaction is repeated once with fresh reagents to ensure completecoupling. The resin is washed three times with methanol and three timeswith dichloromethane.

[0199] Step 2. 4-methoxybenzenesulfonyl chloride (3 eq.) and DIEA (3eq.) are added in dichloromethane to 1 eq. of the resin from Step 1. Thereaction is allowed to proceed for 3 hours, then the reagents aredrained and the resin is washed six times with dichloromethane.

[0200] Step 3 can be accomplished by either of two routes:

[0201] A. 3-(hydroxymethyl) pyridine (1 eq.),tetramethylazodicarboxamide (TMAD) (1 eq.), and triphenylphosphine (1eq.) are added in dichloromethane to 1 eq. of the resin from Step 2. Thereaction is allowed to proceed overnight. The reagents are drained andthe resin is washed six times with dichloromethane.

[0202] B. 3-(bromomethyl) pyridine (2 eq.) and diisopropylethylamine (3eq.) are added in DMF to 1 eq. of the resin from Step 2. The reaction isallowed to proceed overnight. The reagents are drained and the resin iswashed six times with dichloromethane.

[0203] Step 4. The compound is cleaved from the resin by adding 10 ml of95% trifluoroacetic acid/5% anisole to 100 mg of the resin from Step 3.After 60 minutes, the resin is filtered on a sintered glass funnel. Thefiltrate is concentrated to yield the product as a trifluoroacetatesalt.

[0204] Step 5. The product from step 4 is dissolved in methanol. 10%Pd/C is added and the mixture is stirred under a hydrogen atmosphere for5 hours at room temperature. The palladium catalyst is filtered offthrough a layer of Celite, and the filtrate is concentrated to yieldN-hydroxy-2(R)-[[4-methoxybenzenesulfonyl](3-picolyl)amino]-3-methylbutanamide.

Example 6A

[0205]

[0206] The compound depicted above is synthesized by the method ofExample 6, with the exception that Step 3 is omitted.

Example 6B

[0207]

[0208] The compound depicted above is synthesized by using the method ofExample 6 up to and including Step 2. Then NaN(SiCH₃)₂ and benzylbromide are added in dimethylformamide. The reagents are drained andthen Steps 4 and 5 of Example 6 are followed to yield the compounddepicted.

Example 7

[0209]

[0210] Step 1. The resin product of Example 2 (1 eq.) is reacted withsuccinic anhydride (4 eq.) and DIEA (4 eq.) in dichloromethane for 2hours. The reagents are filtered from the resin and the resin is washedthree times with dichloromethane.

[0211] Step 2. The resin produced in Step 1 (1 eq.) is reacted with HBTU(1 eq.), HOBt (1 eq.), and DIEA (1.0 eq.) in DMF for 1 hour. Then thebenzyl ester of L-valine (3 eq.) is added in DMF to the resin suspensionand allowed to react for 1 hour. The reagents are filtered from theresin and the resin is washed three times with DMF, then three timeswith dichloromethane.

[0212] Step 3. The compound is cleaved from the resin by adding 10 ml of95% trifluoroacetic acid/5% anisole to 100 mg of the resin from Step 2.After 60 minutes, the resin is filtered on a sintered glass funnel. Thefiltrate is concentrated to yield the product as a trifluoroacetatesalt.

[0213] Step 4. The product from step 3 is dissolved in methanol. 10%Pd/C is added and the mixture is stirred under a hydrogen atmosphere for5 hours at room temperature. The palladium catalyst is filtered offthrough a layer of Celite, and the filtrate is concentrated to yield thecompound depicted above.

Example 8

[0214]

[0215] Step 1. 2-isobutyl succinic acid monomethyl ester (3 eq.), oxalylchloride (3 eq.), and bis(trimethylsilyl) trifluoroacetamide (3 eq.) arecombined in dichloromethane at 0° C. After 10 minutes the reactionmixture is added to 1 equivalent of the alkoxyamine resin product fromExample 2, and the reaction is allowed to warm to room temperature.After 3 hours the reagents are drained and the resin washed six timeswith dichloromethane.

[0216] Step 2. A solution of 50% 1 M K₂CO₃/50% methanol is added to theresin from Step 1 and allowed to react overnight. The reagents aredrained and the resin washed three times with 50% H₂O/50% methanol, thenthree times with DMF.

[0217] Step 3. A solution of diisopropylcarbodiimide (1 eq.),hydroxybenzotriazole hydrate (1 eq.), and DIEA (1 eq.) in DMF is addedto the resin from Step 2 and allowed to react for 1 hour. Then themethyl ester of L-t-butylglycine (3 eq.) is added in DMF and thereaction is allowed to proceed for one hour. The reagents are removed byfiltration and the resin is washed three times with DMF.

[0218] Step 4. A solution of 50% 1 M K₂CO₃/50% methanol is added to theresin from Step 3 and allowed to react overnight. The reagents aredrained and the resin washed three times with 50% H₂O/50% methanol, thenthree times with DMF.

[0219] Step 5. A solution of 1-hydroxy-7-azabenzotriazole (HOAt) (3eq.), O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), and DIEA (4.5 eq.) is added in DMF to theresin of Step 4 and allowed to react for 1 hour. Then a solution ofaniline (10 eq.) in DMF is added and the reaction allowed to proceedovernight. The reagents are drained and the resin is washed six timeswith DMF, then three times with dichloromethane.

[0220] Step 6. The compound is cleaved from the resin by adding 10 ml of95% trifluoroacetic acid/5% anisole to 100 mg of the resin from Step 5.After 60 minutes, the resin is filtered on a sintered glass funnel. Thefiltrate is concentrated to yield the product as a trifluoroacetatesalt.

[0221] Step 7. The product from step 6 is dissolved in methanol. 10%Pd/C is added and the mixture is stirred under a hydrogen atmosphere for5 hours at room temperature. The palladium catalyst is filtered offthrough a layer of Celite, and the filtrate is concentrated to yield thecompound depicted above.

Example 9 Generation of a Combinatorial Library of N-hydroxamic Acids

[0222] The procedure of Example 7 is followed, with the followingmodifications:

[0223] in Step 1, a mixture of succinic anhydride (1.3 eq.), maleicanhydride (1.3 eq.), and glutaric anhydride (1.3 eq.) is used in placeof the succinic anhydride.

[0224] in Step 2, a mixture of the benzyl esters of all 20 naturallyoccurring L-amino acids is used in place of the benzyl ester ofL-valine. Each amino acid benzyl ester is present in 0.15 eq.

[0225] Steps 3 and 4 are carried out as described in Example 7.

[0226] The result will be a mixture of approximately 60 distinctcompounds (each compound containing one of the three anhydride groupsand one of the 20 amino acids). If desired, an equimolar mixture of thecompounds can be prepared by measuring the kinetics of coupling of thevarious components and adjusting the ratios in solution to ensure equalamounts of coupling. The coupling rates of the various anhydrides can bemeasured by 1) preparing the pure intermediate product of Step 1 foreach anhydride; 2) determining the elution of each pure intermediateusing an analytical method such as HPLC; 3) reacting a mixture ofanhydrides as described in this example, Step 1; 4) determining theratios of the products of Step 1 produced; and 5) adjusting theconcentrations of the anhydrides so that they are present inconcentrations which are inversely proportional to their rate ofcoupling. The coupling of the various amino acids can be determined in asimilar manner, except that acid hydrolysis of the product of Step 4 ofthis example and amino acid analysis is used to determine the relativeamounts of amino acid coupled. Note that tryptophan cannot bequantitated by acid hydrolysis, and asparagine and glutamine will beconverted to aspartate and glutamate, respectively. The concentrationsof these amino acids in the pool of compounds can be assayed by othermethods of amino acid analysis well-known in the art.

Example 10

[0227]

Synthesis of Protected Hydroxylamine—Linker-Resin Compound 53

[0228] O-tetrahydropyranylhydroxylamine (Compound 51) (1.3 g) andCompound 52 (2.6 g, 0.9 equiv.; purchased from PerSeptive Biosystems) inTHF (30 ml) and trimethyl orthoformate (TMOF) (5 ml) were stirred for 2hr at room temperature under nitrogen. Acetic acid (HOAc) (35 μl) and 1M NaCNBH₃ in THF (19.4 ml, 2 equiv.) were added to the mixture andstirred for 18 hr. Solvent was removed under reduced pressure. The crudematerial was loaded on a silica gel column and eluted with DCM-MeOH—HOAc(99-0.15-0.04). Yield: 2.58 g, 63%. ¹H NMR (300 MHz, CDCl₃) δ1.24-1.77(m, 6H), 2.04-2.13 (m, 2H), 2.52-2.57 (t, J=7.14, 2H), 3.53 (m, 2H),3.77 (s, 6H), 3.97-4.01 (t, J=6.17,3H) 4.14 (d, J=2.2, 2H), 4.89 (t,J=2.47, 1H), 6.08 (s, 2H).

Compound 54

[0229] To Compound 53 (1.0 g) and DIEA (0.948 ml, 2 equiv.) in DCM (20ml) was added Fmoc-Cl (0.73g, 1.05 equiv.). The reacture mixture wasstirred for 2 hr at room temperature under argon. Solvent was removedunder vacuum and the crude oil was redissolved in EtOAc (50 ml) andwashed with 0.5 N aqueous HCl (1×50 ml), then H₂O (1×50 ml). The organiclayer was dried with MgSO₄, filtered and solvent removed under vacuum.The crude oil was loaded on a silica gel column and eluted withDCM-MeOH—HOAc (99-0.08-0.02). Yield: 1.42 g, 89%.

Compound 55

[0230] Compound 54 (780 mg, 1.1 equiv.), HATU (502 mg, 1.1 equiv.) andDIEA (694 μl, 3.3 equiv.) were added to TENTAGEL S NH₂ resin (5g, 0.24mmole/g) in DMF (5 ml); the reaction mixture was then shaken for 5 hr.The resin was filtered and washed with MeOH (3×8 ml) and THF (3×8 ml).The resin was dried under vacuum to give compound 55.

Example 11

[0231]

Derivatization of Resin-Bound Protected Hydroxylamine Preparation ofCompound 56

[0232] Compound 55 from Example 10 (300 mg) was treated with 20%piperidine/DMF (5 ml) for 20 min. The resin was washed with MeOH (3×5ml) and DCM (2×5 ml). DIEA (126 μl, 10 equiv.) and hydrocinnamoylchloride (53.4 μl, 5 equiv) in DCM (5 ml) were added at room temperatureunder nitrogen. The reaction mixture was shaken for 18 hr. The resin wasthen washed with MeOH (3×5 ml) and DCM (2×5 ml).

[0233] 2.5% TFA, 1% H₂O in DCM (4 ml) was added to the resin, and thereaction mixture shaken for 1 hr, followed by washes with MeOH (3×5ml)and DCM (2×5ml). 5% H₂O, 50% TFA in DCM was then added to the resin andshaken for 1 hr. The resin was filtered; the filtrate was removed andthe solvent evaporated to give compound 56. ¹H NMR (300 MHz, CDCl₃)δ2.23 (t, 2H), 2.97 (t, 2H), 7.15 (m, 5H).

Example 12

[0234]

Derivatization of Resin-Bound Protected Hydroxylamine Preparation ofCompound 57

[0235] Compound 55 from Example 10 (300 mg) was treated with 20%piperidine/DMF (5 ml) for 20 min. The resin was washed with MeOH (3×5ml) and DCM (2×5 ml). 1 M succinic anhydride in DMF (5 ml) was added tothe resin at room temperature under nitrogen, and the reaction mixtureshaken for 18 hr. The resin was washed with MeOH (3×5 ml) and DCM (2×5ml).

[0236] Benzyl amine (78.6 μl, 10 equiv.) and DIC (113 μl, 10 equiv.)were added to the resin in DCM (5 ml). The reaction mixture was shakenfor 18 hr at room temperature under nitrogen. The resin was washed withMeOH (3×5 ml) and DCM (2×5 ml). 2.5% TFA, 1% H₂O in DCM (4 ml) was addedto the resin, and the reaction mixture shaken for 1 hr, followed bywashes with MeOH (3×5ml) and DCM (2×5 ml).

[0237] 5% H₂O, 50% TFA in DCM was added to the resin and shaken for 1hr. The resin was filtered; the filtrate was removed and evaporated togive compound 57. ¹H NMR (300 5 MHz, CDCl₃) δ3.66 (m, 2H), 4.44 (d, 2H),7.27 (m, 5H).

Example 13

[0238]

Derivatization of Resin-Bound Protected Hydroxylamine Preparation ofCompound 58

[0239] Compound 55 from Example 10 (300 mg) was treated with 20%piperidine/DMF (5 ml) for 20 min. The resin was washed with MeOH (3×5ml) and DCM (2×5 ml). Fmoc—L-Phe (280 mg, 10 equiv.) and DIC (56 μl, 5equiv.) were added to the resin in DMF (5 ml) at room temperature undernitrogen. The reaction mixture was shaken for 18 hr.

[0240] The resin was washed with MeOH (3×5ml) and DCM (2×5ml).

[0241] 20% Piperidine/DMF (5 ml) was added to the resin and the reactionmixture shaken for 20 min. The resin was washed with MeOH (3×5 ml) andDCM (2×5 ml).

[0242] Pyridine (2 ml) and acetic anhydride (1 ml) were added to theresin and shaken for 2 hr. The resin was washed with MeOH (3×5 ml) andDCM (2×5 ml).

[0243] 2.5% TFA, 1% H₂O in DCM (4 ml) was added to the resin, and thereaction mixture shaken for 1 hr and washed with MeOH (3×5 ml) and DCM(2×5 ml).

[0244] 5% H₂O, 50% TFA in DCM was added to the resin and shaken for 1hr. The resin was filtered; the filtrate was removed and evaporated togive compound 58. ¹H NMR (300 MHz, CDCl₃) δ1.91 (s, 3H), 3.03 (m, 2H),4.59 (m, 1H), 7.27 (m, 5H).

Example 14 Synthesis of Compound 63 (see FIG. 5)

[0245] O-allylhydroxyamine (Compound 61) (637 mg) and Compound 62 (2.6g,0.9 equiv.) in THF (30 ml) and trimethyl orthoformate (TMOF) (5 ml) werestirred for 2 hr at room temperature under nitrogen. Acetic acid (35 μl)and 1 M NaCNBH₃ in THF (19.4 ml, 2 equiv.) were added to the mixture andstirred for 18 hr. Solvent was removed under reduced pressure. The crudematerial was loaded on a silica gel column and eluted with DCM-MeOH-AcOH(99-0.15-0.04). Yield: 2.2 g, 70%. ¹H NMR: (300 MHz, CDCl₃) δ2.03-2.10(m, 1H), 2.21-2.26 (m, 1H), 2.45-2.50 (t, J=7.55 Hz, 2H), 3.77 (s, 3H),3.81 (s, 3H), 3.96-4.00 (t, J=6.32 Hz, 1H), 4.10-4.14 (t, J=5.22 Hz,1H), 4.40 (s, (dd, J=6.32 Hz, J=4.26 Hz, 2H), 5.39-5.45 (m, 2H),5.76-5.90 (m, 1H), 6.05-611 (m 2H).

Example 15 Synthesis of Compound 64 (see FIG. 5)

[0246] To Compound 63 (1.0 g) and DIEA (1.07 ml, 2 equiv.) in DCM (20ml) was added Fmoc-Cl (0.835 g, 1.05 equiv.), and the reaction mixturewas stirred for 2 hr at room temperature under argon. Solvent wasremoved under vacuum and the crude oil was redissolved in EtOAc (50 ml)and washed with 0.5 N aqueous HC1 (1×50 ml) and H₂O(1×50 ml). Theorganic layer was dried with MgSO₄, filtered and the solvent removedunder vacuum. The crude oil was loaded on a silica gel column and elutedwith DCM-MeOH—HOAc (99-0.08-0.02). Yield: 1.51 g, 89%. ¹H NMR: (300 MHz,CDCl₃) 6 2.034-2.099 (m, 1H), 2.09-2.15 (m, 2H), 2.55-2.62 (m, 2H),3.76-3.81 (t, J=6.87 Hz, 6H), 3.96-13 (m, 3H), 4.26-4.31 (m, 1H),4.46-4.51 (t, J=7.28 Hz, 2H), 4.79 (d, J=3.85 Hz, 2H), 5,06-5.16 (m,2H), 5.62-5.79 (m, 1H), 6.09 (m, 2H), 7.26-7.41 (m, 4H), 7.65-7.76 (m,4H).

Example 16 Synthesis of Compound 65 (see FIG. 5)

[0247] Compound 64 (655 mg, 1.1 equiv.), HATU (502 mg, 1.1 equiv.) andDIEA (694 μl, 3.3 equiv.) were added to TENTAGEL S NH₂ resin (5 g, 0.24mmole/g) in DMF (5 ml), and the reaction mixture shaken for 5 hr. Theresin was filtered and washed with MeOH (3×8 ml) and THF (3×8 ml). Theresin was dried under vacuum to give Compound 65.

Example 17 Synthesis of Compound 56 (see FIG. 6)

[0248] 20% piperidine in DMF (5 ml) was added to Compound 65 (300 mg)and the resin shaken for 20 minutes. The resin was washed with MeOH (3×5ml) and DCM (2×5 ml). DIEA (126 μl, 10 equiv.) and hydrocinnamoylchloride (53.4 μl, 5 equiv.) in DCM (5 ml) were added at roomtemperature under nitrogen. The reaction mixture was shaken for 18 hr.The resin was washed with MeOH (3×5 ml) and DCM (2×5 ml). Then 1% H₂O,50% TFA in DCM was added to the resin and the resin shaken for 1 hr. Theresin was filtered and the solvent was removed from the filtrate toyield Compound 66. ¹H NMR: (300 MHz, CDCl₃) 6 2.19 (br-s, 2H), 2.97 (t,2H), 4.36 (br-s, 2H), 5.27 (m, 2H), 1H), 7.18 (m, 5H).

[0249] To Compound 66 (100 mg) in acetonitrile (2 ml) was addedtetrakis(triphenylphosphine)palladium(0) (11.6 mg), triphenylphosphine(5.3 mg), and pyrrolidine (50 μl) at room temperature under argon. Thereaction was stirred for 18 hr., the solvent was removed under reducedpressure, and the crude material was purified by preparatory TLC (5%MeOH in DCM). ¹H NMR: (300 MHz, CDCl₃) 6 2.23 (t, 2H), 2.97 (t, 2H),7.15 (m, 5H).

Example 18 Synthesis of Compound 70 (see FIG. 7)

[0250] Compound 55 from Example 10 (300 mg) was shaken with 20%piperidine in DMF (5 ml) for 20 min. The resin was washed with MeOH (3×5ml) and DCM (2×5 ml). Fmoc-D-Valine (203 mg, 10 equiv.) and DIC(diisopropylcarbodiimide) (47 μl, 5 equiv.) were added to the resin inDMF (5 ml) at room temperature under nitrogen. The reaction mixture wasshaken for 18 hr. The resin was washed with MeOH (3×5 ml) and DCM (2×5ml) to yield Compound 68.

[0251] Compound 68 (300 mg) was shaken with 20% piperidine in DMF (5 ml)for 20 minutes. The resin was washed with MeOH (3×5 ml) and DCM (2×5ml). Then N-methylmorpholine (132 μl, 20 equiv.) and4-methoxybenzenesulfonyl chloride (124 mg, 10 equiv.) in DCM were addedto the resin at room temperature and the reaction mixture shaken for 4hr. The resin was washed with MeOH (3×5 ml) and DCM (2×5 ml) to yieldCompound 69.

[0252]2.5% TFA, 1% H₂O in DCM (4 ml) was added to the resin and thereaction mixture shaken for 1 hr, followed by washing with MeOH (3×5 ml)and DCM (2×5 ml).

[0253] Then 1% H₂O, 50% TFA in DCM was added to the resin and the resinshaken for 1 hr. The resin was filtered and the solvent was removed fromthe filtrate to yield Compound 70. ¹HNMR: (300 MHz, CDCl₃) δ1.05 (d,6H), 2.18 (m, 1H), 3.81 (s, 3H), 4.29 (m, 1H), 6.98 (d, 2H), 7.77 (d,2H).

Example 19 Synthesis of Compound 71 (see FIG. 8)

[0254] To Compound 69 (300 mg) in THF (5 ml) was added 1 M sodiumbis(trimethylsilyl)amide in THF (1.2 ml, 20 equiv.) and benzyl bromide(14823 μl, 20 equiv.) at room temperature under argon. The reactionmixture was shaken for 18 hr. The resin was washed with MeOH (3×5 ml)and DCM (2×5 ml). 2.5% TFA, 1% H₂O in DCM (4 ml) was added to the resinand the reaction mixture shaken for 1 hr, followed by washing with MeOH(3×5 ml) and DCM (2×5 ml). Then 1% H₂O, 50% TFA in DCM was added to theresin and the resin shaken for 1 hr. The resin was filtered and thesolvent was removed from the filtrate to yield Compound 71. ¹H NMR: (300MHz, CDCl₃) δ1.05 (d, 6H), 2.18 (m, 1H), 3.81 (s, 3H), 4.29 (m, 1H),5.07 (s, 2H), 6.98 (d, 2H)

Example 20 Synthesis of Compound 72 (see FIG. 9)

[0255] To Compound 69 (300 mg) in THF (4 ml) was addedtriphenylphosphine (524 mg, 0.5 M), pyridylcarbinol (388 μl, 1.0 M), anddiisopropylazodicarboxylate (394 μl, 0.5 M) at room temperature underargon. The reaction was shaken for 18 hr. The resin was washed with MeOH(3×5 ml) and DCM (2×5 ml). 2.5% TFA, 1% H₂O in DCM (4 ml) was added tothe resin and the reaction mixture shaken for 1 hr, followed by washingwith MeOH (3×5 ml) and DCM (2×5 ml). Then 1% H₂O, 50% TFA in DCM wasadded to the resin and the resin shaken for 1 hr. The resin was filteredand the solvent was removed from the filtrate to yield Compound 72.

Example 21 Synthesis of Compound 73 (see FIG. 10)

[0256] To Compound 68 (300 mg) was added 20% piperidine in DMF (5 ml).The resin was shaken for 20 minutes, then washed with MeOH (3×5 ml) andDCM (2×5 ml). Pyridinecarboxaldehyde (113 μl, 20 equiv.) in trimethylorthoformate (5 ml) was stirred for 30 min. at room temperature undernitrogen. HOAc (100 μl, 2%) and 1 M NaCNBH₃ in THF (1.8 ml, 30 equiv.)were added to the reaction mixture and stirred for 18 hr. The resin waswashed with MeOH (3×5 ml) and DCM (2×5 ml).

[0257] N-methylmorpholine (132 μl, 20 equiv.) and 4-methoxybenzenesulfonyl chloride (124 mg, 10 equiv.) were added to the resin at roomtemperature. The reaction was shaken for 4 hr. and the resin washed withMeOH (3×5 ml) and DCM (2×5 ml).

[0258] 2.5% TFA, 1% H₂O in DCM (4 ml) was added to the resin and thereaction mixture shaken for 1 hr, followed by washing with MeOH (3×5 ml)and DCM (2×5 ml). Then 1% H₂O, 50% TFA in DCM was added to the resin andthe resin shaken for 1 hr. The resin was filtered and the solvent wasremoved from the filtrate to yield Compound 73.

[0259] Note that as Compound 72 and Compound 73 are identical, the aboveExample 21 illustrates an alternate synthetic route to the synthesisused in Example 20.

Example 22 Use of O-Allyl Protected Hydroxylamine Resin in Solid-phaseSynthesis and Solid-Phase Combinatorial Synthesis

[0260] The O-allyl protected hydroxylamine resin (Compound 65) can bederivatized in analogous fashion as the O-THP protected resin in theprevious examples. However, after derivatization is complete, thecompounds are cleaved from the resin by using 1% H₂O, 50% TFA in DCM.The O-allyl protecting group is not removed by this procedure. To removethe O-allyl protecting group, Pd(PPh₃)₄/PPh₃/pyrrolidine in acetonitrileis used (where PPh₃ is triphenylphosphine).

[0261] Alternatively, the allyl group can be removed while the compoundsare still attached to the resin, by treating the resin withPd(PPh₃)₄/PPh₃/pyrrolidine in acetonitrile. The compounds can then beremoved from the resin by using 1% H20, 50% TFA in DCM.

Example 23 Synthesis of a Combinatorial Library of HydroxylamineCompounds

[0262]

[0263] Generation of a combinatorial library can be accomplished by thereaction scheme outline above, either by 1) utilizing a mixture of aminoacid reagents at each step in the synthesis; 2) using the “split andpool” method to react resin portions with different reagents andrecombining; or 3) synthesizing small number of compounds separately,then combining the compounds at the end of the procedure to form thelibrary. While the reaction scheme depicts amino acids, imino acidsincluding, but not limited to, Fmoc—L-proline and Fmoc-D-proline can beused as well.

[0264] N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, SanDiego, Calif.) (100 mg, 0.75 mmol/g) is shaken for 2 hr. in a solutionof 20% piperidine in DMF (2 ml). The reaction is washed with MeOH (2×2ml) and DCM (3×2 ml). Then the Fmoc amino acid, Fmoc-NH—CH(R,)—COOH (10equiv) and DIC (5 equiv) are added to the resin in DMF (2 ml), and thereaction mixture is shaken for 18 hr. The reaction mixture is washedwith MeOH (2×2 ml) and DCM (3×2 ml). A solution of 20% piperidine in DMF(2 ml) is added to the resin and shaken for 45 min.; the resin is thenwashed with MeOH (2×2 ml) and DCM (3×2 ml). Then the Fmoc amino acidFmoc-NH—CH(R₂)-COOH (10 equiv) and DIC (5 equiv) are added to the resinin DMF (2 ml), and the reaction mixture shaken for 18 hr. The reactionmixture is washed with MeOH (2×2 ml) and DCM (3×2 ml). A solution of 20%piperidine in DMF (2 ml) is added to the resin and shaken for 20 min;the resin is then washed with MeOH (2×2 ml) and DCM (3×2 ml). R₃COOH (10equiv) and DIC (5 equiv) are added to the resin in DMF (2 ml), and thereaction mixture shaken for 18 hr. The reaction mixture is washed withMeOH (2×2 ml) and DCM (3×2 ml). A solution of 10% TFA, 5% TES in DCM isadded to reaction and shaken for 30 min, filtered, and the filtrate isconcentrated under reduced pressure.

Example 24

[0265]

[0266] The sulfonamide nitrogen of the compounds in the figure above canbe derivatized by using Route B (which attaches a pyridylmethyl group)or Route C (which attaches a methyl group), or left underivatized byusing Route A. Route B is described in Steps 3 and 4 of Example 6. RouteA is described in Step 4 of Example 6. Route C is described as follows:iodomethane (10 equiv) was added to a solution oft-butylimino-tri(pyrrolidino) phosphorane (phosphazene baseP₁-t-Bu-tris(tetramethylene), Fluka, Ronkonkoma, N.Y.) (20 equiv) andresin (100 mg) in DMF (2 ml). The reaction mixture was shaken for 18 hr.and the resin was filtered and washed sequentially with MeOH (3×5 ml)and DCM (2×5 ml). A solution of 2.5% TFA and 1% H₂O in DCM (4 ml) wasthen added, and the mixture was shaken for 1 h. The resin was filteredand washed sequentially with MeOH (3×5 ml) and DCM (2×5 ml). A solutionof 50% TFA and 1% H₂O in DCM (4 ml) was then added, the mixture shakenfor 1 h, filtered, and the filtrate was concentrated under reducedpressure.

[0267] By utilizing various reagents for Fmoc—NH-CHR₁ -COOH andR₂—SO₂—Cl, and by utilizing Routes A, B and C as described above, acombinatorial library can be synthesized.

Example 25

[0268]

[0269] Synthesis of a Combinatorial Library of Hydroxamic AcidsContaining a Hydantoin Group.

[0270] A suspension of resin (0.05 mmol N-Fmoc-hydroxylamine2-chlorotrityl resin, Novabiochem, San Diego, Calif.) is deprotectedwith 20% piperidine in DMF. The resin is washed with MeOH (3×5 ml) andDMF (3×5 ml). To the resulting resin is added Fmoc—NH—CHR₁—COOH (5 eq.),DIEA (15 eq.) and PyBOP (5 eq.). The reaction mixture is shaken for twohours, then the resin is drained and washed with MeOH (3×5 ml) and DMF(3×5 ml).

[0271] The resin is then deprotected with 20% piperidine in DMF. Theresin is washed with MeOH (3×5 ml) and DMF (3×5 ml). To the resultingresin is added Fmoc—NH—CHR₂—COOH (5 eq.), DIEA (15 eq.) and PyBOP (5eq.). The reaction mixture is shaken for two hours, then the resin isdrained and washed with MeOH (3×5 ml) and DMF (3×5 ml).

[0272] The resin is then deprotected with 20% piperidine in DMF. Theresin is washed with MeOH (3×5 ml) and DMF (3×5 ml).

[0273] DIEA (10 equiv.) in THF is then added to the resin. Subsequently,4-nitrophenyl chloroformate (p-NP-Cl) (5 eq.) is added. The reactionmixture is shaken for 18 hours at RT; then the resin is washed with DMF(2×5 mL) and DCM (3×5 ml).

[0274] A solution of 10% DIEA in DMF is added to the resin and themixture is heated at 80° C. overnight. Then the resin is washed withMeOH (2×5 ml) and DCM (3×5 ml).

[0275] A solution of 10% TFA and 5% triethylsilane (TES) in DCM (4 mL)is then added to the resin and the reaction mixture is shaken for 30minutes. The resin is filtered; the filtrate is collected and thesolvent evaporated under reduced pressure to yield the producthydantoin.

[0276] By introducing a variety of amino acids at the steps whereFmoc—NH—CHR₁—COOH and Fmoc-NH—CHR₂—COOH are coupled to the resin (forexample, by using a cocktail of amino acids at each step; by splittingthe resin, reacting single amino acids separately, and recombining theresin; or by synthesizing compound discretely and mixing them together)a combinatorial library of hydroxylamine compounds containing ahydantoin moiety is synthesized.

[0277] All references, publications and patents mentioned herein arehereby incorporated by reference herein in their entirety.

[0278] Although the forgoing invention has been described in some detailby way of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practical. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A library comprising a plurality of hydroxylamine and/orhydroxylamine derivative compounds, wherein the library is prepared bypreparing a solid support-bound alkoxyamine; derivatizing the solidsupport bound alkoxyamine; cleaving the derivatized alkoxylamines fromthe solid support; and removing the alkoxyl protecting group.
 2. Thelibrary of claim 1 , where the step of preparing a solid support-boundalkoxyamine comprises adding an alkoxylamine nucleophile comprising analkoxy protecting group to a solid support comprising a leaving group,thereby displacing the leaving group from the solid support to produce asolid support-bound alkoxyamine.
 3. The library of claim 1 , where thestep of preparing a solid support-bound alkoxyamine comprises adding analkoxy-protected hydroxylamine-linker intermediate comprising anO-protected alkoxyamine and a linker group to a solid support bearing anamine group to produce a solid support-bound alkoxyamine.
 4. The libraryof claim 1 , wherein the hydroxylamine and/or hydroxylamine derivativecontaining compounds are selected from the group consisting ofhydroxylamines, hydroxamic acids, hydroxyl ureas, andhydroxylsulfonamides.
 5. The library of claim 1 , wherein thehydroxylamine and/or hydroxylamine derivative containing compounds arehydroxamic acids.
 6. The library of claim 1 , wherein the hydroxylamineand/or hydroxylamine derivative containing compounds are hydroxyl ureas.7. A library comprising a plurality of hydroxylamine and/orhydroxylamine derivative compounds, wherein the library contains atleast 40 distinct compounds.
 8. The library of claim 7 , wherein the atleast 40 distinct compounds are selected from compounds of the formulas

wherein the R groups are independently selected from the groupconsisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclicmoieties and naturally and non-naturally occurring amino acid sidechains; m, n, and x are integers independently selected from 0 to 12;and y is an integer selected from 0 to 30, and all stereoisomers,protected derivatives, and salts thereof.
 9. The library of claim 7 ,wherein the at least 40 distinct compounds are selected from compoundsof the formula L₃—L₂—L₁—NHOH, where L₃ is selected from the groupconsisting of

L₂ is selected from the group consisting of

and all stereoisomers, protected derivatives, and salts thereof.
 10. Thelibrary of claim 7 , wherein the at least 40 distinct compounds areselected from compounds of the formula L₁₂—S(═O)₂—L₁₁—NHOH, where L₁₂ isselected from the group consisting of

and L₁₁ is selected from the group consisting of

and all stereoisomers, protected derivatives, and salts thereof.
 11. Thelibrary of claim 7 , wherein the at least 40 distinct compounds areselected from compounds of the formula L₂₂—S(═O)₂—L₂₁—NHOH where L₂₂ isselected from the group consisting of

and L₂₁ is selected from the group consisting of

where Z is

or —CH; and all stereoisomers, protected derivatives, and salts thereof.12. The library of claim 7 , wherein the at least 40 distinct compoundsare selected from compounds of the formula:

where R₁ is selected from the group consisting of

and L is selected from the group consisting of

and all stereoisomers, protected derivatives and salts thereof.
 13. Thelibrary of claim 7 , wherein the at least 40 distinct compounds areselected from compounds of the formula L₂—L₁—NHOH where L₂ is selectedfrom the group consisting of —H, —C(═O)—CH₃, —C(═O)—OCH₃, and—C(═O)—CH₂—C(═O)—OH; and L₁ is selected from the group consisting of

and all stereoisomers, protected derivatives and salts thereof.
 14. Amethod for the synthesis of a library comprising a plurality ofhydroxylamine and/or hydroxylamine derivative compounds, comprising thesteps of preparing a solid support-bound alkoxyamine; derivatizing thesolid support bound alkoxyamine; cleaving the derivatized alkoxylaminesfrom the solid support; and removing the alkoxyl protecting group. 15.The method of claim 14 , where the step of preparing a solidsupport-bound alkoxyamine comprises adding an alkoxylamine nucleophilecomprising an alkoxy protecting group to a solid support comprising aleaving group, thereby displacing the leaving group from the solidsupport to produce a solid support-bound alkoxyamine.
 16. The library ofclaim 14 , where the step of preparing a solid support-bound alkoxyaminecomprises adding an alkoxy-protected hydroxylamine-linker intermediatecomprising an O-protected alkoxyamine and a linker group to a solidsupport bearing an amine group to produce a solid support-boundalkoxyamine.
 17. The method of claim 14 , wherein the hydroxylamine andhydroxylamine derivative containing compounds are selected from thegroup consisting of hydroxylamines, hydroxamic acids, hydroxyl ureas,and hydroxylsulfonamides.
 18. The method of claim 14 , wherein thehydroxylamine and hydroxylamine derivative containing compounds arehydroxamic acids.
 19. The method of claim 14 , wherein the hydroxylamineand hydroxylamine derivative containing compounds are hydroxyl ureas.20. The method of claim 14 , wherein the alkoxylamine nucleophilecomprising an alkoxy protecting group is selected from the groupconsisting of O-trityl hydroxylamine, O-(t-butyldimethylsilyl)hydroxylamine, O-allyl hydroxylamine, O-benzyl hydroxylamine,O-(4-methoxybenzyl) hydroxylamine, O-(2,4-dimethoxybenzyl)hydroxylamine, and O-(2-tetrahydropyranyl) hydroxylamine.
 21. The methodof claim 14 , wherein the leaving group is selected from the groupconsisting of bromide, iodide, and mesylate.
 22. The method of claim 14, wherein the solid support comprising a leaving group isbromomethylphenoxy resin.
 23. An O-protected hydroxylaminefunctionalized resin, suitable for the preparation of a librarycontaining hydroxylamine and/or hydroxylamine derivative compounds. 24.The O-protected hydroxylamine functionalized resin of claim 23 ,prepared by adding an alkoxylamine nucleophile comprising an alkoxyprotecting group to a solid support comprising a leaving group, therebydisplacing the leaving group from the solid support to produce a solidsupport bound alkoxyamine.
 25. The O-protected hydroxylaminefunctionalized resin of claim 23 , prepared by adding a protectedhydroxylamine-linker intermediate comprising an O-protected alkoxyamineand a linker group to a solid support bearing an amine group to producea solid support-bound alkoxyamine.
 26. The O-protected hydroxylaminefunctionalized resin of claim 24 , wherein the alkoxylamine nucleophilecomprising an alkoxy protecting group is selected from the groupconsisting of O-trityl hydroxylamine, O-(t-butyldimethylsilyl)hydroxylamine, O-allyl hydroxylamine, O-benzyl hydroxylamine,O-(4-methoxybenzyl) hydroxylamine, O-(2,4-dimethoxybenzyl) hydroxylamineand O-(2-tetrahydropyranyl) hydroxylamine.
 27. The O-protectedhydroxylamine functionalized resin of claim 24 , wherein thealkoxylamine nucleophile is selected from the group consisting ofO-allyl hydroxylamine and O-(2-tetrahydropyranyl) hydroxylamine.
 28. TheO-protected hydroxylamine functionalized resin of claim 24 , wherein theleaving group is selected from the group consisting of bromide, iodide,and mesylate.
 29. The O-protected hydroxylamine functionalized resin ofclaim 25 , wherein the O-protected alkoxyamine is selected from thegroup consisting of O-trityl hydroxylamine, O-(t-butyldimethylsilyl)hydroxylamine, O-allyl hydroxylamine, O-benzyl hydroxylamine,O-(4-methoxybenzyl) hydroxylamine, O-(2,4-dimethoxybenzyl) hydroxylamineand O-(2-tetrahydropyranyl) hydroxylamine.
 30. The functionalized resinof claim 25 , wherein the alkoxylamine nucleophile is selected from thegroup consisting of O-allyl hydroxylamine and O-(2-tetrahydropyranyl)hydroxylamine.
 31. A composition of the formula

wherein b is an integer from 1 to 5, J is —OH or —NH—RESIN, and Q is —Sor —T, where —S is a leaving group selected from the group consisting ofbromide, iodide, mesylate, tosylate, and p-nitrophenylsulfonate, and —Tis —NHOP₁ where P₁ is a protecting group selected from the groupconsisting of 2-tetrahydropyranyl, trityl, t-butyldimethylsilyl, allyl,benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, andRESIN is any solid or polymeric support.
 32. The composition of claim 31, where RESIN is a polyethylene glycol-polystyrene graft copolymerresin.
 33. An O-protected hydroxylamine-linker compound suitable forattachment to an amine-bearing resin, comprising a cleavable linkergroup and an O-protected hydroxylamine, wherein the linker group isacid-labile and/or photolabile.
 34. The O-protected hydroxylamine-linkercompound of claim 33 , wherein the linker group is acid-labile. 35.O-protected hydroxylamine-linker compounds according to claim 34 ,wherein the O-protected hydroxylamine-linker compounds are representedby the formula

where b is an integer from 1 to 5, P₁ is a protecting group selectedfrom the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and2,4-dimethoxybenzyl protecting groups, J₂ is —H or —Fmoc, and J₁ is —OHor —NH-RESIN, where RESIN is any solid or polymeric support.
 36. TheO-protected hydroxylamine-linker compounds of claim 35 , where J₁ is—NH-RESIN and RESIN is a polystyrene-polyethylene glycol graft copolymerresin.
 37. The O-protected hydroxylamine-linker compounds of claim 35 ,where P₁ is allyl.
 38. The O-protected hydroxylamine-linker compounds ofclaim 35 , where P₁ is 2-tetrahydropyranyl.
 39. The O-protectedhydroxylamine-linker compound of claim 33 , wherein the linker group isphotolabile.
 40. O-protected hydroxylamine-linker compounds according toclaim 39 , wherein the O-protected hydroxylamine-linker compounds arerepresented by the formula:

where b is an integer from 1 to 5, P₁ is a protecting group selectedfrom the group consisting of 2-tetrahydropyranyl, trityl,t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and2,4-dimethoxybenzyl protecting groups, J₂ is —H or —Fmoc, and J₁ is —OHor —NH-RESIN, where RESIN is any solid or polymeric support.
 41. Aderivatized resin, where the resin is selected from the group consistingof hydroxymethylphenoxy resin and 2-methoxy-4-alkoxybenzyl alcoholresin, wherein the active hydroxyl group of the resin is replaced with aleaving group selected from the group consisting of bromide, iodide,mesylate, tosylate, and p-nitrophenylsulfonate.
 42. The derivatizedresin of claim 41 , wherein the active hydroxyl group of the resin isreplaced with a leaving group selected from the group consisting ofbromide and iodide.